The consequences of acute cold exposure on protein oxidation and proteasome activity in short-tailed field voles,microtus agrestis

During cold exposure, animals upregulate their metabolism and food intake, potentially exposing them to elevated reactive oxygen species (ROS) production and oxidative damage. We investigated whether acute cold (7 +/- 3 degrees C) exposure (1, 10, or 100 h duration) affected protein oxidation and proteasome activity, when compared to warm controls (22 +/- 3 degrees C), in a small mammal model, the short-tailed field vole Microtus agrestis. Protein carbonyls and the chymotrypsin-like proteasome activity were measured in plasma, heart, liver, kidney, small intestine (duodenum), skeletal muscle (gastrocnemius), and brown adipose tissue (BAT). Trypsin-like and peptidyl-glutamyl-like proteasome activities were determined in BAT, liver, and skeletal muscle. Resting metabolic rate increased significantly with duration of cold exposure. In skeletal muscle (SM) and liver, protein carbonyl levels also increased with duration of cold exposure, but this pattern was not repeated in BAT where protein carbonyls were not significantly elevated. Chymotrpsin-like proteasome activity did not differ significantly in any tissue. However, trypsin-like activity in SM and peptidyl-glutamyl-like activity in both skeletal muscle and liver, were reduced during the early phase of cold exposure (1-10 h), correlated with the increased carbonyl levels in these tissues. In contrast there was no reduction in proteasome activity in BAT during the early phase of cold exposure and peptidyl-glutamyl-like activity was significantly increased, correlated with the lack of accumulation of protein carbonyls in this tissue. The upregulation of proteasome activity in BAT may protect this tissue from accumulated oxidative damage to proteins. This protection may be a very important factor in sustaining uncoupled respiration, which underpins nonshivering thermogenesis at cold temperatures.     

p130/p107 expression distinguishes adipogenic potential in primary myoblasts based on age

Recent investigations have provided significant evidence that many mesodermally derived tissues contain stem cell-like precursors capable of being stimulated to undergo differentiation into a variety of cellular lineages. We have recently reported that primary myoblasts isolated from 23-month-old mice have an increased adipogenic potential when compared to their 8-month-old counterparts. To further characterize the degree of adipocyte differentiation in these myoblasts, we examined early and late markers of adipocyte differentiation. Within the first 24h of adipocyte differentiation, expression of p130 and p107, two members of the retinoblastoma tumor suppressor gene family, are regulated and this event is an important one early in adipogenesis. Consistent with the increased adipogenic potential of the older myoblasts and in contrast to the younger cells, the p130:p107 pattern of expression is very similar to that observed in adipogenesis where there is a transient increase in p107 expression accompanied by a decrease in p130 expression. Interestingly, while these older cells accumulated lipid and expressed genes associated with lipid metabolism, they failed to express adipsin and leptin, two well-established markers of terminal adipocyte differentiation. These results suggest that older myoblasts are capable of initiating and progressing through the adipogenic program to a point where they express genes associated with lipid metabolism, but do not reach a terminally differentiated state. This finding may have important metabolic implications in the aging population.     

Bovine blastocyst development from oocytes injected with freeze-dried spermatozoa

Pronuclear formation, and the chromosomal constitution and developmental capacity of bovine zygotes formed by intracytoplasmic sperm injection with freeze-dried (lyophilized) spermatozoa were evaluated. Frozen-thawed spermatozoa were selected, freeze-dried, and stored at 4 degrees C until use. After 22-24 h of in vitro maturation oocytes were denuded and injected singly with a lyophilized spermatozoon. Injected oocytes were activated by treatment with 10 microM ionomycin (5 min) alone and in combination with 1.9 mM 6-dimethylaminopurine (DMAP) for 4 h. Ionomycin plus DMAP activation treatment resulted in a significantly higher proportion of sperm-injected oocytes with two pronuclei than was found after activation with ionomycin alone (74% vs. 56%; P < 0.03). The rates of cleavage, morula, and blastocyst development of sperm-injected oocytes treated with ionomycin plus DMAP were higher than after activation with ionomycin alone (63.3%, 34.2%, and 29.6% vs. 44.7%, 18.7%, and 10.6%, respectively; P < 0.05). Seventy-three percent of blastocysts produced with lyophilized sperm were diploid. These results demonstrate that in vitro-matured bovine oocytes can be fertilized with freeze-dried sperm cells, and that resultant zygotes can develop into karyotypically normal blastocysts.     

Modulation of the onset of postnatal development of H(+)-ATPase-rich cells by steroid hormones in rat epididymis

Vacuolar type H(+)-ATPase is involved in lumenal acidification of the epididymis. This protein is highly expressed in narrow and clear cells where it is located in the apical pole, and it contributes to proton secretion into the lumen. We have previously shown that in rats, epididymal cells rich in H(+)ATPase appear during postnatal development and reach maximal numbers at 3-4 wk of age. The factors that regulate the appearance of these cells have not been investigated, but androgens, estrogens, or both may be involved. This study examined whether neonatal administration of estrogens (diethylstilbestrol [DES] or ethinyl estradiol) or an antiandrogen (flutamide), or the suppression of androgen production via administration of a GnRH antagonist (GnRHa), was able to alter the appearance of cells rich in H(+)-ATPase in the rat epididymis when assessed at age 25 days. Surprisingly, all of these treatments were able to significantly reduce the number of H(+)-ATPase positive cells; this was determined by immunofluorescence and confirmed by Western blotting. In contrast, neonatal coadministration of DES and testosterone maintained the expression of H(+)-ATPase in the epididymis at Day 25 despite the high level of concomitant estrogen exposure. These findings indicate that androgens, acting via the androgen receptor, are essential for the normal development of epididymal cells rich in H(+)-ATPase, and that treatments that interfere directly or indirectly with androgen production (GnRHa, DES) or action (flutamide, DES) will result in reduced expression of H(+)-ATPase. Our findings do not exclude the possibility that estrogens can directly suppress the postnatal development of cells in the epididymis that are rich in H(+)-ATPase, but if this is the case, this suppression can be prevented by testosterone administration.     

Optical and IR absorption as probe of dynamics of heme proteins

The spectroscopy of horseradish peroxidase with and without the substrate analogue benzohydroxamic acid (BHA) was monitored in different solvents as a function of the temperature in the interval from 10 to 300 K. Thermal broadening of the Q(0,0) optical absorption band arises mainly from interaction of the electronic pi --> pi transition with the heme vibrations. In contrast, the width of the IR absorption band of CO bound to heme is controlled by the coupling of the CO transition moment to the electric field of the protein matrix. The IR bandwidth of the substrate free enzyme in the glycerol/H2O solvent hardly changes in the glassy matrix and strongly increases upon heating above the glass transition. Heating of the same enzyme in the trehalose/H2O glass considerably broadens the band. The binding of the substrate strongly diminishes the temperature broadening of the CO band. This result is consistent with the view that the BHA strongly reduces the amplitude of vibrations of the heme pocket environment. Unusually strong thermal broadening of the CO band above the glass transition is interpreted to be caused by thermal population of a very flexible excited conformational substate. The thermal broadening of the same band in the trehalose glass is caused by an increase of the protein vibrational amplitude in each of the conformational substates, their population being independent of the temperature in the glassy matrix.     

Probing protein-DNA interactions by unzipping a single DNA double helix

We present unzipping force analysis of protein association (UFAPA) as a novel and versatile method for detection of the position and dynamic nature of protein-DNA interactions. A single DNA double helix was unzipped in the presence of DNA-binding proteins using a feedback-enhanced optical trap. When the unzipping fork in a DNA reached a bound protein molecule we observed a dramatic increase in the tension in the DNA, followed by a sudden tension reduction. Analysis of the unzipping force throughout an unbinding "event" revealed information about the spatial location and dynamic nature of the protein-DNA complex. The capacity of UFAPA to spatially locate protein-DNA interactions is demonstrated by noncatalytic restriction mapping on a 4-kb DNA with three restriction enzymes (BsoBI, XhoI, and EcoRI). A restriction map for a given restriction enzyme was generated with an accuracy of approximately 25 bp. UFAPA also allows direct determination of the site-specific equilibrium association constant (K(A)) for a DNA-binding protein. This capability is demonstrated by measuring the cation concentration dependence of K(A) for EcoRI binding. The measured values are in good agreement with previous measurements of K(A) over an intermediate range of cation concentration. These results demonstrate the potential utility of UFAPA for future studies of site-specific protein-DNA interactions.     

Molecular basis of the voltage-dependent gating of TREK-1, a mechano-sensitive K(+) channel

TREK-1 is a member of the mammalian two P domain K(+) channel family. Mouse TREK-1 activity, in transiently transfected COS cells, is reduced at negative resting membrane potentials by both an external Mg(2+) block and an intrinsic voltage-dependent gating mechanism leading to a strong outward rectification. Deletional and chimeric analysis demonstrates that the carboxy terminal domain of TREK-1, but not the PKA phosphorylation site S333, is responsible for voltage-dependent gating. Since the same region is also critically required for TREK-1 mechano-gating, both mechanisms might be functionally linked. Preferential opening of TREK-1 at depolarized potentials will greatly affect action potential duration, recovery from inactivation and neuronal repetitive firing activity.     

Regulation of T cell receptor-induced activation of the Ras-ERK pathway by diacylglycerol kinase zeta

T cell development in the thymus and activation of mature T cells in the periphery depend on signals stimulated by engagement of the T cell antigen receptor (TCR). Among the second messenger cascades initiated by TCR ligation include the phosphatidylinositol pathway where the membrane phospholipid, phosphatidylinositol 4,5-bisphosphate, is hydrolyzed to inositol 1,4,5-trisphosphate and diacylglycerol (DAG). Inositol 1,4,5-trisphosphate signals a rise in intracellular free calcium, leading to translocation of nuclear factor of activated T cells into the nucleus. DAG activates RasGRP and protein kinase C theta. Because both RasGRP and protein kinase C theta are essential for thymocyte and T cell function, it is critical to understand how DAG is regulated. In this report, we demonstrate expression of DAG kinase zeta (DGKzeta, the enzyme that catalyzes the conversion of DAG to phosphatidic acid) in multiple lymphoid organs, with highest expression observed within the T cell compartment. Overexpression studies in Jurkat T cells indicate that DGKzeta interferes with TCR-induced Ras and ERK activation, AP-1 induction, and expression of the activation marker CD69. In contrast, TCR-stimulated calcium influx is not altered. Mutational analysis indicates that the kinase and DAG binding domains, but not the ankyrin repeats of DGKzeta, are required for its inhibitory effects. Collectively these studies demonstrate a potential role of DGKzeta to function as a selective negative regulator of DAG signaling on T cell activation and provide the first structure/function analysis of this enzyme in T cells.